Sc 48357

Liver tissues or hepatocytes lysates were extracted with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, 50 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate 8.0) including 0.5% protease inhibitors and 1% phosphorylation inhibitors. The protein samples were loaded into a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane. After 5% bovine serum albumin (BSA) blocked for 1 h, the membrane was incubated with primary antibodies at 4 °C overnight, and then secondary antibodies for 1 h at room temperature. Immunoreactivities were detected with enhanced chemiluminescent autoradiography (Merck Millipore, Billerica, MA, USA). Chemiluminescence was determined using the AI600 System (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The antibodies used for immunoblotting specific to SREBP1 (1:1000, 14,088–1-AP, Proteintech), FASN (1:1000, SC-48357, Santa Cruz), carnitine palmitoyl transferase 1 alpha (CPT1α, 1:1000, 15,184–1-AP, Proteintech), SCD1 (1:1000, SC-515875, Santa Cruz), GAPDH (1:1000, 60,004–1-Ig, Proteintech), α-Actinin (1:1000, Cat No. 11313–2-AP, Proteintech).

Cells were treated with 40 μM of RSV with or without ALA for 24 h. For protein analysis, whole cell protein lysates from HepG2 were harvested with RIPA buffer (50 mmol/l Tris, 150 mmol/l NaCl, 0.5% sodium deoxycholate (v/v), 0.1% SDS (w/v), 1% NP-40 (v/v), pH 7.4) supplemented with proteinase inhibitors. Protein concentrations were determined with the Pierce™ BCA assay (Thermo Fisher Scientific GmbH, life technologies™, Darmstadt, Germany) according to the manufacturer's instructions and analyzed by Western Blotting analysis as described before (40 (link)). Briefly, 30 μg per samples were heated with loading buffer and applied to Mini-PROTEAN® Stainfree™ Precast Gels (4–20%, BioRad laboratories GmbH, Munich, Germany). Samples were transferred to a polyvinylidenedifluoride membrane (Bio-Rad laboratories GmbH, Munich, Germany) and blocked with skim milk dissolved in Tris-buffered saline + 0.05% (v/v) Tween 20. Membranes were then incubated with the primary antibodies FADS1, FADS2, CPT1, GAPDH (sc-134337, sc-98480, sc-48357, and sc-20357, respectively; Santa Cruz Biotechnology Inc., Dallas, USA) and FASN (bs-5045R, Bioss Inc., Massachusetts, USA). Secondary antibodies were from Santa Cruz Biotechnology Inc., Dallas, USA. Results were analyzed using Image Lab 5.0 (Bio-Rad laboratories GmbH, Munich, Germany).